Molecular display technologies have been developed,to construct and screen polypeptide and antibody display libraries and to develop recombinant vaccines, adsorbents, recombinant biocatalysts, and solid phase reagents for diagnostic and analytical purposes. The concept of molecular display technology is the provision of a physical linkage between genotype (e.g., antibody variable region genes) and phenotype (e.g., antigen-binding) to allow simultaneous selection of the genes that encode a protein with the desired function (e.g., binding).
Phage display has been used to probe polypeptide-ligand interactions and extensively for the isolation of high-affinity proteins, including antibodies (Winter G, Milstein C. 1991. Man-made antibodies. Nature 349: 293–299; Vaughan T. J., Williams A. J., Pritchard K., Osbourn J. K., Pope A. R., Earnshaw J. C., McCafferty J., Hodits R. A., Wilton J., Johnson K. S. 1996. Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat Biotechnol 14: 309–314). Phage libraries are typically screened using repeated cycles of phage capture and elution from an immobilized ligand followed by phage amplification in bacteria. A display technology wherein polypeptides are physically linked in vitro to their coding RNA through a ribosome was recently described (Hanes J., Pluckthun A. 1997. In vitro selection and evolution of functional proteins by using ribosome display. Proc Natl Acad Sci USA 94: 4937–42). However, both phage and ribosome used in these two technologies cannot self-replicate and are too small to be assisted by the powered cell sorting technology, such as fluorescence-activated cell sorting (FACS).
FACS can facilitate high throughput and quantitative screening of protein or antibody libraries displayed on the surface of bacteria, because the relatively large sizes of bacterial cells can allow the system to pick up the clones that bind to fluorescently labeled ligands with high affinity and specificity (Daugherty P S, Olsen M J, Iverson B L, Georgiou G. 1999. Development of an optimized expression system for the screening of antibody libraries displayed on the Escherichia coli surface. Protein Eng. 12: 613–21; Steidler L; Viaene J; Fiers W; Remaut E. 1996. Functional display of a heterologous protein on the surface of Lactococcus lactis by means of the cell wall anchor of Staphylococcus aureus protein A. Immunotechnology. 2: 97–102; Andreoni C, Goetsch L, Libon C, Samuelson P, Nguyen T N, Robert A, Uhlen M, Binz H, Stahl S. 1997. Flow cytometric quantification of surface-displayed recombinant receptors on staphylococci. Biotechniques. 23: 696–702, 704). With current high-speed cell sorters, 108 cells can be screened quantitatively in 1 h, a throughput similar to that obtained in phage library selections. Thus, cell surface display coupled with FACS provides an important alternative display technology for protein engineering. Phage display formats designed for sensitive affinity selections rely upon a single molecular binding event or monovalent display through gpIII fusions. In contrast, cell surface formats achieve typically 104–105 copies per cell. Consequently, stochastic variations in stability or expression level do not interfere with cell surface library selections.
Bacterial surface display systems have been developed since small peptides fused onto some outer membrane proteins, such as OmpA, LamB, and PhoE, were found to be directed to the Escherichia coli cell surface (Charbit A, Boulain J. C, Ryter A, Hofnung M. 1986. Probing the topology of a bacterial membrane protein by genetic insertion of a foreign epitope: expression at the cell surface. EMBO J. 5: 3029–37; Freudl R, MacIntyre S, Degen M, Henning U. 1986. Cell surface exposure of the outer membrane protein OmpA of Escherichia coli K-12. J Mol Biol. 188: 491–494; Agterberg M, Adriaanse H, Tommassen J. 1987. Use of outer membrane protein PhoE as a carrier for the transport of a foreign antigenic determinant to the cell surface of Escherichia coli K-12. Gene. 59: 145–50). One of the most intriguing applications of expressing proteins on bacterial cell surface is to develop live bacterial vaccines. Presentation of antigens on bacterial cell surface is considered to be advantageous since the surface-exposed antigens might be better recognized by the immune systems. Moreover, the outer membrane lipopolysaccharides can enhance the immune response and may serve as an adjuvant for surface-anchored polypeptides (Lee J. S., Shin K. S., Pan J. G., and Kim C. J. 2000. Surface-displayed viral antigens on Salmonella carrier vaccine. Nat. Biotechnol. 18: 645–648). Further, bacteria are easy to be manipulated and inexpensive to be manufactured and distributed. Successful examples of producing new recombinant vaccines have been reported (Kim E J; Yoo S K. 1999. Cell surface display of hepatitis B virus surface antigen by using Pseudomonas syringae ice nucleation protein. Lett Appl Microbiol 29: 292–297; Lee J. S., Shin K. S., Pan J. G., and Kim C. J. 2000. Nat. Biotechnol. 18: 645–648). Other applications of bacterial surface display systems include the production of whole-cell adsorbents for bioremediation and novel biocatalysts (Kim Y S.; Jung H C; Pan J G. 2000. Bacterial cell surface display of an enzyme library for selective screening of improved cellulase variants. Appl Environ Microbiol. 66: 788–793; Jung H C; Lebeault J M; Pan J G. 1998. Surface display of Zymomonas mobilis levansucrase by using the ice-nucleation protein of Pseudomonas syringae. Nat Biotechnol. 16: 576–80).
The commonly used Lpp-OmpA surface display system includes three parts: the signal sequence and first nine N-terminal amino acids of the mature major E. coli lipoprotein (Lpp), amino acids 46–159 of E. coli outer membrane protein OmpA, and the passenger protein (Francisco J A, Earhart C F, Georgiou G. 1992. Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci USA. 89: 2713–2717). This system has been successfully used to display β-lactamase (Francisco J A, Earhart C F, Georgiou G. 1992. Proc Natl Acad Sci USA. 89: 2713–2717), green fluorescent protein (Shi, Huidong; Su, Wei Wen. 2001. Display of green fluorescent protein on Escherichia coli cell surface. Enzyme and Microbial Technology. 28: 25–34), single chain Fv (scFv) libraries (Daugherty P S; Chen G; Olsen M J; Iverson B L, Georgiou G. 1998. Antibody affinity maturation using bacterial surface display. Protein Eng. 11: 825–32; Daugherty P S, Olsen M J, Iverson B L, Georgiou G. 1999. Development of an optimized expression system for the screening of antibody libraries displayed on the Escherichia coli surface. Protein Eng. 12: 613–21) and recently the HIV-reverse transcriptase (Burnett M S, Wang N. Hofmann M, Kitto G B. 2001. Potential live vaccines for HIV. Vaccine. 19: 735–742). However, this system has limitations: (1) The cell viability is much lower when the Lpp-OmpA is under a constitutive expression condition; thus it is critical to use a tightly regulatory system and an appropriate plasmid of low copy number to optimize the display, especially for large passenger proteins (Daugherty P S, Olsen M J, Iverson B L, Georgiou G. 1999. Development of an optimized expression system for the screening of antibody libraries displayed on the Escherichia coli surface. Protein Eng. 12: 613–21; Earhart C. F. 2000. Use of an Lpp-OmpA fusion vehicle for bacterial surface display. Methods Enzymol. 326: 506–516). (2) This system cannot display a dimeric protein like bacterial alkaline phosphatase (PhoA) (Stathopoulos C, Georgiou G, Earhart C F. 1996. Characterization of Escherichia coli expressing an Lpp'OmpA(46–159)-PhoA fusion protein localized in the outer membrane. Appl Microbiol Biotechnol. 45: 112–119). The function of PhoA requires the formation of disulfide bridges, which is not spontaneous but catalyzed at least at the disulfide bond formation step (Bardwell J C, McGovern K, Beckwith J. 1991. Identification of a protein required for disulfide bond formation in vivo. Cell 67: 581–589; Kamitani S, Akiyama Y, Ito K. 1992. Identification and characterization of an Escherichia coli gene required for the formation of correctly folded alkaline phosphatase, a periplasmic enzyme. EMBO J 11: 57–62).
Many active proteins and enzymes are of multi-subunit nature; and many require disulfide bridges for intra- and inter-molecular interactions. The formation of disulfide bonds and protein folding of passenger proteins in the periplasmic space can hinder the successful display of these proteins. Thus, reducing agents have been added into the culturing medium to increase the efficiency of translocation of CtxB domain and PhoA (Klauser T, Pohlner J, Meyer T F. 1990. Extracellular transport of cholera toxin B subunit using Neisseria IgA protease beta-domain: conformation-dependent outer membrane translocation. EMBO J. 9: 1991–1999; Stathopoulos et al., 1996; Suzuki T, Lett M C, Sasakawa C. 1995. Extracellular transport of VirG protein in Shigella. J Biol Chem. 270: 30874–30880). However, addition of reducing agents in the growth medium can break up the-disulfide bonds required for intra- and inter-molecular interactions. This may interfere with the activity and/or display of some enzymes or proteins; it may also alter the bacterial growth and gene expression, in addition to increasing the operation costs, particularly for large-scale applications.
References relevant to cell surface display technology include: U.S. Pat. No. 5,348,867, U.S. Pat. No. 5,516,637, U.S. Pat. No. 5,866,344, U.S. Pat. No. 6,274,345, U.S. Pat. No. 6,300,065, and U.S. Pat. No. 6,190,662.